Expression of DDX25 in nuage components of mammalian spermatogenic cells: immunofluorescence and immunoelectron microscopic study

Abstract

The localization of DDX25/GRTH and gonadotropin-stimulated RNA helicase was studied in the spermatogenic cells of rat, mouse, and guinea pig by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence studies identified four kinds of granular staining: (1) fine particles observed in meiotic cells; (2) small granules associated with a mitochondrial marker, appearing in pachytene spermatocytes after stage V; (3) short strands lacking the mitochondrial marker in late spermatocytes; and, (4) large irregularly shaped granules in round spermatids. IEM identified DDX25 signals in nine compartments: (1) fine dense particles in the meiotic cells; (2) intermitochondrial cement; (3) loose aggregates of 70-90 nm particles; (4) chromatoid bodies; (5) late chromatoid bodies; (6) satellite bodies; (7) granulated bodies; (8) mitochondria-associated granules; and, (9) reticulated bodies. Compartments (1) to (6) were previously classified into nuage while (7) to (9) were classified as nuage components by the present study. The results suggest that DDX25 functions in these nine compartments.

Expression and Localization of NANOS1 in Spermatogenic Cells during Spermatogenesis in Rat

“…The NANOS1 signals were weaker than those observed for other nuage proteins such as DDX4 [23], DDX25 [21] and BRU-NOL2 [22]. As shown by immunofluorescence staining and dot blotting analysis, NANOS1 was abundantly present in the cytosol fraction.…”

“…The present study showed that NANOS1 is also localized to the non-nuage structures, such as MAG, GB, RB, RA, Head cap of the step-17 spermatids, and outer dense fibers of flagella. Although the functions of MAG, GB, RB, and RA remain unknown, our previous studies have shown that DDX25 and BRUNOL2 are present in these structures [21,23]. The existence of nuage proteins such as BRUNOL2, DDX25 and NANOS1 in these structures suggests that these proteins may have biological functions there.…”

“…Despite of the colocali-zation of NANOS1 with DDX4 in the CB, the interaction of the two proteins has not been shown in other nuage compartments such as intermitochondrial cement (IMC) and irregularly-shaped perinuclear granules (ISPG). Moreover, it has been reported that several nuage-resident proteins are localized to the non-nuage structures, including mitochondria-associated granules (MAG), granulated body (GB), reticulated body (RB) and ribosome aggregates (RA) [21,22]. It is unclear whether NANOS1 is localized to the nuage or non-nuage compartments.…”

PRKRA Localizes to Nuage Structures and the Ectoplasmic Specialization and Tubulobulbar Complexes in Rat and Mouse Testis

“…From these studies a new step of nuage research was started. Previously, we investigated the localization of several nuage-resident proteins in the nuage structures of spermatogenic cells by immunoelectron microscopy (IEM) [22][23][24][25][26]. On the basis of localization of nuage proteins, including DDX4, DDX25, BRUNOL2, NANOS1, and MAEL, we reclassified the nuage into (1) aggregates of 60-90 nm particles (60-90P); (2) irregularly-shaped perinuclear granules (ISPG); (3) intermitochondrial cement (IMC); (4) satellite bodies (SB); and (5) chromatoid bodies (CB) [25].…”

“…Cluster of 30 nm particles which were classified into the nuage by Russell and Frank [19] were not nuage because they did not contain any nuage proteins. In addition, we determined that several nuage proteins localize to spermatogenic cell-specific structures, including mitochondria-associated granules (MAG), 2 Journal of Histology granulated bodies (GB), reticulated bodies (RB), and ribosome aggregates (RA) [22][23][24][25][26]. These structures first appear in the cytoplasmic lobe of elongated step 10 spermatids and subsequently disappear in step 19 spermatids.…”